Leucine-rich repeats 2-4 (Leu60-Glu128) of platelet glycoprotein Ibα regulate shear-dependent cell adhesion to von Willebrand factor

Glycoprotein (GP) Ib-IX-V binds von Willebrand factor (VWF), initiating thrombosis at high shear stress. The VWF-A1 domain binds the N-terminal domain of GPIb alpha (His(1)-Glu(282)); this region contains seven leucine-rich repeats (LRR) plus N- and C-terminal flanking sequences and an anionic sequence containing three sulfated tyrosines. Our previous analysis of canine/human and human/canine chimeras of GPIb alpha expressed on Chinese hamster ovary (CHO) cells demonstrated that LRR2-4(Leu(60)-Glu(128)) were crucial for GPIb alpha-dependent adhesion to VWF. Paradoxically, co-crystal structures of the GPIb alpha N-terminal domain and GPIb alpha-binding VWF-A1 under static conditions revealed that the LRR2-4 sequence made minimal contact with VWF-A1. To resolve the specific functional role of LRR2-4, we compared wildtype human GPIb alpha with human GPIb alpha containing a homology domain swap of canine for human sequence within Leu(60)-Glu(128) and a reverse swap (canine GPIb alpha with human Leu(60)-Glu(128)) for the ability to support adhesion to VWF under flow. Binding of conformation-specific anti-GPIb alpha antibodies and VWF binding in the presence of botrocetin (which does not discriminate between species) confirmed equivalent expression of wild-type and mutant receptors in a functional form competent to bind ligand. Compared with CHO cells expressing wild-type GPIb alpha, cells expressing GPIb alpha, where human Leu(60)-Glu(128) sequence was replaced by canine sequence, supported adhesion to VWF at low shear rates but became increasingly ineffective as shear increased from 50 to 2000 s-(1). Together, these data demonstrate that LRR2-4, encompassing a pronounced negative charge patch on human GPIb alpha, is essential for GPIb alpha(VWF)-V-.-dependent adhesion as hydrodynamic shear increases.